Potassium channel modulators

ABSTRACT

Provided herein are compounds of the formula: 
                                           
or pharmaceutically acceptable salts thereof, and compositions comprising such compounds for use in the treatment of diseases or conditions responsive to modulation of the small conductance calcium-activated potassium channel (SK channel).

RELATED APPLICATIONS

This application is a continuation of U.S. Pat. No. 15,877,910, filedJan. 23, 2018, which claims priority to U.S. Provisional Application No.62/449,270, filed Jan. 23, 2017, the entire contents of each of whichare incorporated herein by reference.

BACKGROUND

Among the ion channels, potassium channels are the most prevalent anddiverse, being found in a variety of animal cells such as nervous,muscular, glandular, immune, reproductive, and epithelial tissue. Thesechannels allow the flow of potassium in and/or out of the cell undercertain conditions. These channels are regulated, e.g., by calciumsensitivity, voltage-gating, second messengers, extracellular ligands,and ATP-sensitivity.

Dysfunction of potassium channels and dysfunction from other causeswhich influence these potassium channels are known to generate loss ofcellular control, altered physiological function, and diseaseconditions. Because of their ability to modulate ion channel functionand/or regain ion channel activity, potassium channel modulators arebeing used in the pharmacological treatment of a wide range ofpathological diseases and have the potential to address an even widervariety of therapeutic indications.

The small conductance calcium-activated potassium channels (SK channel)are a subfamily of Ca²⁺-activated K⁺ channels and the SK channel familycontains 4 members—SK1, SK2, SK3, and SK4 (often referred to asintermediate conductance). The physiological roles of the SK channelshave been especially studied in the nervous system, where for examplethey are key regulators of neuronal excitability and of neurotransmitterrelease, and in smooth muscle, where they are crucial in modulating thetone of vascular, broncho-tracheal, urethral, uterine orgastro-intestinal musculature.

Given these implications, small molecule modulators of potassium ionchannels could have the potential to treat a large variety of diseasescharacterized by dysfunction of potassium ion channels and dysfunctionfrom other causes which influence these potassium channels.

SUMMARY

Disclosed are compounds and pharmaceutically acceptable salts thereof,and pharmaceutical compositions thereof, which are useful in thetreatment of diseases associated with the dysfunction of potassium ionchannels and dysfunction from other causes which influence thesepotassium channels. (See e.g., Table 1).

The compounds described herein were found to have one or more of thefollowing beneficial properties: high solubility, high brain freefraction, little or no hERG inhibition, extended in vivo half-lives,good bioavailability, high liver microsomal stability, enhancedpermeability such as parallel artificial membrane permeability (PAMPA),and/or low Cyp inhibition. See e.g., the comparative data in Tables 2and 3.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram illustrating the effect of Compound 1 following oral(PO) dosing on harmaline induced tremor.

FIG. 2 displays the efficacy and dose response of Compound 1 in percentmotion power.

FIG. 3 shows the effects of Compound 1 on Purkinje cell firingirregularity in ex vivo slices from SCA2 58Q transgenic mice.

FIG. 4 shows the effects of Compound 1 on baseline ataxia in the EA2Tottering mouse model.

DETAILED DESCRIPTION 1. Compounds

Provided herein are compounds of the formula:

or pharmaceutically acceptable salts thereof.

2. Definitions

As used herein the terms “subject” and “patient” may be usedinterchangeably, and means a mammal in need of treatment, e.g.,companion animals (e.g., dogs, cats, and the like), farm animals (e.g.,cows, pigs, horses, sheep, goats and the like) and laboratory animals(e.g., rats, mice, guinea pigs and the like). Typically, the subject isa human in need of treatment.

When the stereochemistry of a disclosed compound is named or depicted bystructure, the named or depicted stereoisomer is at least 60%, 70%, 80%,90%, 99% or 99.9% by weight pure relative to all of the otherstereoisomers. Percent by weight pure relative to all of the otherstereoisomers is the ratio of the weight of one stereoisiomer over theweight of the other stereoisomers. When a single enantiomer is named ordepicted by structure, the depicted or named enantiomer is at least 60%,70%, 80%, 90%, 99% or 99.9% by weight optically pure. Percent opticalpurity by weight is the ratio of the weight of the enantiomer over theweight of the enantiomer plus the weight of its optical isomer.

When a disclosed compound is named or depicted by structure withoutindicating the stereochemistry, and the compound has one chiral center,it is to be understood that the name or structure encompasses oneenantiomer of compound free from the corresponding optical and geometricisomer, a racemic mixture of the compound, and mixtures enriched in oneenantiomer relative to its corresponding optical isomer.

Pharmaceutically acceptable salts as well as the neutral forms of thecompounds described herein are included. For use in medicines, the saltsof the compounds refer to non-toxic “pharmaceutically acceptable salts.”Pharmaceutically acceptable salt forms include pharmaceuticallyacceptable acidic/anionic or basic/cationic salts. Pharmaceuticallyacceptable basic/cationic salts include, the sodium, potassium, calcium,magnesium, diethanolamine, n-methyl-D-glucamine, L-lysine, L-arginine,ammonium, ethanolamine, piperazine and triethanolamine salts.Pharmaceutically acceptable acidic/anionic salts include, e.g., theacetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, carbonate,citrate, dihydrochloride, gluconate, glutamate, glycollylarsanilate,hexylresorcinate, hydrobromide, hydrochloride, malate, maleate,malonate, mesylate, nitrate, salicylate, stearate, succinate, sulfate,tartrate, and tosylate.

The term “pharmaceutically acceptable carrier, adjuvant, or vehicle”refers to a non-toxic carrier, adjuvant, or vehicle that does notdestroy the pharmacological activity of the compound with which it isformulated. Pharmaceutically acceptable carriers, adjuvants or vehiclesthat may be used in the compositions described herein include, but arenot limited to, ion exchangers, alumina, aluminum stearate, lecithin,serum proteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

The terms “treatment,” “treat,” and “treating” refer to reversing,alleviating, reducing the likelihood of developing, or inhibiting theprogress of a disease or disorder, or one or more symptoms thereof, asdescribed herein. In some embodiments, treatment may be administeredafter one or more symptoms have developed, i.e., therapeutic treatment.In other embodiments, treatment may be administered in the absence ofsymptoms. For example, treatment may be administered to a susceptibleindividual prior to the onset of symptoms (e.g., in light of a historyof symptoms and/or in light of genetic or other susceptibility factors),i.e., prophylactic treatment. Treatment may also be continued aftersymptoms have resolved, for example to prevent or delay theirrecurrence.

The term “effective amount” or “therapeutically effective amount”includes an amount of a compound described herein that will elicit abiological or medical response of a subject.

3. Uses, Formulation and Administration

In some embodiments, compounds and compositions described herein areuseful in treating diseases and/or disorders associated with theactivity of potassium channels. Such diseases and/or disorders includee.g., neurodegenerative and neurological conditions (e.g., Parkinson'sdisease, tremors, Alzheimer's disease, dementia, amyotrophic lateralsclerosis (ALS) ataxia, anxiety, depression, mood disorders, memory andattention deficits, bipolar disorder, psychosis, schizophrenia,traumatic brain injury, and narcolepsy), heart disease and relatedconditions (e.g., ischaemic heart disease, coronary heart disease,angina pectoris, and coronary artery spasms), metabolic disease andbladder diseases (e.g., bladder spasms, urinary incontinence, bladderoutflow obstruction, gastrointestinal dysfunction, irritable bowelsyndrome, and diabetes), withdrawal symptoms associated with terminationof addiction, and other conditions associated with the modulation ofpotassium channels such as e.g., respiratory diseases, epilepsy,convulsions, seizures, absence seizures, vascular spasms, renaldisorders (e.g., polycystic kidney disease), erectile dysfunction,secretory diarrhoea, ischaemia, cerebral ischaemia, dysmenorrhea,Reynaud's disease, intermittent claudication, Sjorgren's syndrome,arrhythmia, hypertension, myotonic muscle dystrophia, spasticity,xerostomi, hyperinsulinemia, premature labor, baldness, cancer, immunesuppression, migraine and pain.

The present disclosure also provides a method of modulating the activityof a potassium channel in a subject comprising the step of administeringa compound described herein. In another embodiment, the presentdisclosure provides a method of positively modulating a SK2 channel in acell comprising the step of contacting the cell with a compounddescribed herein.

In one aspect, the provided compounds and compositions are used to treattremors. Tremors include, but are not limited to rest, active, postural,kinetic, intention, task specific, and idiopathic tremors. In oneaspect, the provided compounds and compositions are used to treatpostural and active tremors. Examples of postural and/or active tremorsinclude essential tremor, drug-induced parkinsonism, neuropathic tremor,and tremors induced from toxins (e.g., alcohol withdrawal or fromexposure to heavy metals). In one aspect, the provided compounds andcompositions are used to treat essential tremor.

The present disclosure further provides a method of treating essentialtremor in a subject comprising the step of administering a compound orpharmaceutically acceptable salt or composition described herein.

Essential tremor is one of the most common neurological disorders,affecting ˜0.9% of the general population. Essential tremor ischaracterized by an action tremor of the upper limbs and, less commonly,the head, voice, and trunk. A family history of essential tremor can beidentified in approximately half of patients, suggesting a geneticcomponent. Drinking alcohol often temporarily reduces tremor.

In some embodiments, the present disclosure provides a method oftreating a disease or condition selected from a neurodegenerativedisease, dementia, heart disease, withdrawal symptoms associated withtermination of addiction, metabolic disease, and bladder disease. Inother embodiments, the present disclosure provides a method of treatinga disease or condition selected from ataxia, dystonia, Parkinson'sdisease, ischemia, traumatic brain injury, amyotrophic lateralsclerosis, hypertension, atherosclerosis, diabetes, arrhythmia,over-active bladder, and withdrawal symptoms caused by the terminationof abuse of alcohol and other drugs of abuse. In some embodiments, thepresent disclosure provides a method of treating ataxia. In someembodiments, the present disclosure provides a method of treatingspinocerebellar ataxia.

The present disclosure provides pharmaceutically acceptable compositionscomprising a compound described herein; and a pharmaceuticallyacceptable carrier. These compositions can be used to treat one or moreof the diseases and conditions described above.

Compositions described herein may be administered orally, parenterally,by inhalation spray, topically, rectally, nasally, buccally, vaginallyor via an implanted reservoir. The term “parenteral” as used hereinincludes subcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intralesionaland intracranial injection or infusion techniques. Liquid dosage forms,injectable preparations, solid dispersion forms, and dosage forms fortopical or transdermal administration of a compound are included herein.

The amount of provided compounds that may be combined with carriermaterials to produce a composition in a single dosage form will varydepending upon the patient to be treated and the particular mode ofadministration. In some embodiments, provided compositions may beformulated so that a dosage of between 0.01-100 mg/kg body weight/day ofthe provided compound, such as e.g., 0.1-100 mg/kg body weight/day, canbe administered to a patient receiving these compositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including age, body weight, general health, sex, diet, time ofadministration, rate of excretion, drug combination, the judgment of thetreating physician, and the severity of the particular disease beingtreated. The amount of a provided compound in the composition will alsodepend upon the particular compound in the composition.

Exemplification

The representative examples that follow are intended to help illustratethe present disclosure, and are not intended to, nor should they beconstrued to, limit the scope of the invention.

N-(4,4-difluorocyclohexyl)-2-(3-methyl-1H-pyrazol-1-yl)-6-morpholinopyrimidin-4-amine

Step 1:

A round-bottomed flask equipped with a teflon-coated stir bar wascharged with 4,6-dichloro-2-(methylsulfonyl)pyrimidine (20.0 g, 88.080mmol, 1.0 eq) in tetrahydrofuran at −10° C. and 3-methyl-1H-pyrazole(7.23 g, 88.080 mmol, 1.0 equiv.) was added dropwise over a period offive minutes via syringe. The reaction mixture was stirred for 16 hoursat 25° C. and completion of reaction was determined by TLC. The reactionmixture was portioned between water (500 mL) and ethyl acetate (500 mL).The organic layer was separated and the aqueous layer was extracted withethyl acetate (2*100 mL). The combined organic layer was dried oversodium sulfate, filtered, and concentrated under reduced pressure toafford crude product which was purified by column chromatography (ethylacetate/hexane as solvent system) to afford4,6-dichloro-2-(3-methyl-1H-pyrazol-1-yl)pyrimidine (10.0 g, 43.859mmol, 50% yield) as a white solid pure form. MS (MH+): m/z=229.1.

Step 2:

A round-bottomed flask equipped with a teflon-coated stir bar wascharged with 2,4-dichloro-6-methylpyrimidine (11.0 g, 48.24 mmol, 1.0equiv.), 4,4-difluorocyclohexan-1-amine hydrochloride (9.89 g, 57.89mmol, 1.2 equiv.), and Cs₂CO₃ (39.19 g, 120.61 mmol, 2.5 equiv.) inacetonitrile (200 mL). The reaction mixture was stirred for five hoursat 80° C. and the completion of reaction was determined by TLC. Thereaction mixture was cooled to room temperature and partitioned betweenwater (100 mL) and ethyl acetate (200 mL). The organic layer wasseparated and the aqueous layer was extracted ethyl acetate (2×100 mL).The combined organic layer was dried over sodium sulfate, filtered, andconcentrated under reduced pressure to afford crude product which waspurified by column chromatography (ethyl acetate/hexane as solventsystem) to afford6-chloro-N-(4,4-difluorocyclohexyl)-2-(3-methyl-1H-pyrazol-1-yl)pyrimidin-4-amine(11.0 g, 33.62 mmol, 71%) as an off-white solid. MS (MH+): m/z=328.1.

Step 3:

A round-bottomed flask equipped with a teflon-coated stir bar wascharged with6-chloro-N-(4,4-difluorocyclohexyl)-2-(3-methyl-1H-pyrazol-1-yl)pyrimidin-4-amine(14.0 g, 42.79 mmol, 1.0 eq), morpholine (14.91 mL, 171.19 mmol, 4.0eq), and triethylamine (23.89 mL, 171.19 mmol, 4.0 eq) in acetonitrile(200 mL). The reaction mixture was stirred for 16 hours at 80° C. andcompletion of reaction was determined by TLC. The reaction mixture wascooled to room temperature and partitioned between water (100 mL) andethyl acetate (300 mL). The organic layer was separated and the aqueouslayer was extracted ethyl acetate (2×100 mL). The combined organic layerwas dried over sodium sulfate, filtered, and concentrated under reducedpressure to afford crude product which was purified by columnchromatography (ethyl acetate/hexane as solvent system) to affordN-(4,4-difluorocyclohexyl)-2-(3-methyl-1H-pyrazol-1-yl)-6-morpholinopyrimidin-4-amine(1) (12.8 g, 33.84 mmol, 79% yield) as an off-white solid.

Analytical Data:

MS (MH⁺): m/z=379.2; ¹H NMR (400 MHz, DMSO-D6): δ 8.41 (d, J=2 Hz, 1H),7.07 (d, J=8.3 Hz, 1H), 6.25 (d, J=2.4 Hz, 1H), 5.53 (s, 1H), 3.9 (bs,1H), 3.67 (t, J=4.4 Hz, 4H), 3.49 (S, 4H), 2.23 (s, 3H), 2.23-1.97 (m,3H), 1.92-1.90 (m, 3H), 1.55-1.53 (m, 2H). ¹H NMR exhibited, viaintegration, 3 proton resonances in the aromatic region and 1 broadresonance corresponding to the exchangeable proton at N-18. The aromaticprotons were observed as two doublets and a singlet, indicating twoadjacent protons, and one isolated aromatic proton. In the aliphaticregion, resonances corresponding to 20 protons were observed, showing 1distinct singlet. Integration of these resonances corresponded to twoupfield doublet-doublet resonances, one with a partially overlappingmultiplet proton, and four downfield multiplets, two of which partiallyoverlap. The upfield aliphatic multiplets are associated with themorpholine moiety of the structure. The downfield multiplets are splitadditionally by the proximity of CF₂ to these protons. In the highresolution (HR) LC/MS analysis, the pseudomolecular ion (M+H⁺) wasobserved at low fragmentor voltage (70V) in ESI positive ion mode at m/z379.20580. The other prominent ion observed at m/z 779.38575 isattributable to the in-source dimer adduct 2M+Na⁺.

¹³C NMR data revealed 14 separate carbon resonances and were generatedfrom a decoupled ¹³C carbon spectrum acquired at 25° C. in CDCl₃. The 14resonances are consistent with the structure of 1 in which four pairs ofthe 18 carbons are spectroscopically equivalent. One carbon resonance(C-12 at 77.05 ppm) was observed to be partially obscured by the CHCl₃resonance in the spectrum. The presence of a carbon at this resonancewas confirmed by collection of a ¹³C spectrum in C₆D₆; a resonance at79.7 ppm was observed without interference from the solvent peak, inaddition to all other resonances observed. A triplet (2J=244 Hz) isobserved at the C-22 resonance of 122.29, two additional triplets areobserved at 31.53 ppm (C-21 & C-23, 3J=24.93 Hz), and at 31.53 ppm (C-20& C-24, 4J=5.37 Hz). Each of the triplets observed are consistent withF2 substitution at C-22, and decreasing coupling constant with respectto fluorine as the number of intervening bonds increase.

Carbon-proton connectivity, and carbon-carbon connectivity (via 2 and3-bond C—C—H correlations) of the molecular framework was confirmed bythe collection of 2D NMR spectra. Direct C—H connectivity was confirmedby HSQC, and 2- and 3-bond connectivity was demonstrated by HMBC. Theshort- and long-range cross correlations (over 2 or 3 bonds) areconsistent with the connections expected from the proposed structure.Finally, observed chemical shift data assigned as shown above wasconsistent with computer-predicted ¹H and ¹³C chemical shifts for theproposed structure.

N-(4,4-difluorocyclohexyl)-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-methoxypyrimidin-4-amine

Step 1:

A 5000-mL four-necked, flame-dried, round-bottomed flask, equipped witha teflon-coated stir blade (5 cm) attached with glass rod (neck 1),stopper (neck 2), and addition funnel with stopper (neck 3) and anitrogen gas inlet-outlet U-tube adaptor filled with oil (Neck 4), wascharged with a suspension of sodium hydride (35.2 g, 880 mmol, 1 equiv.)in dichloromethane (1000 mL) was added 3,5-dimethylpyrazole (84.6 g, 880mmol, 1 equiv.) at 0° C. and the reaction mixture was stirred at roomtemperature. After 30 min, 4,6-dichloro-2-(methylsulfonyl)pyrimidine(200 g, 880 mmol, 1 equiv.) (dissolved in dichloromethane (1000 mL)) wasadded dropwise through dropping funnel to the reaction mixture at −78°C. The reaction mixture was stirred at same temperature and thecompletion of reaction was determined by TLC and UPLC. After 2 h, thereaction mixture was quenched with water at −78° C. and diluted withdichloromethane. After 5 min, dichloromethane was decanted and washedwith brine solution. The organic layer was dried over sodium sulfate,filtered, and concentrated under reduced pressure to afford crudeproduct, which was purified by column chromatography using ethyl acetateand pet-ether as solvent to afford 4,6-dichloro-2-(3,5-dimethyl-1h-pyrazol-1-yl) pyrimidine (138 g, 567.71 mmol, 65%) as anoff-white solid. MS (MH+): m/z=244.2.

Step 2:

A 2000-mL three-necked, flame-dried, round-bottomed flask, equipped witha teflon-coated stir bar (5 cm), one septa (neck 1), stopper (neck 3)and reflux condenser equipped with nitrogen gas inlet-outlet U-tubeadaptor filled with oil (Neck 2), was charged with a solution of4,6-dichloro-2-(3,5-dimethyl-1h-pyrazol-1-yl) pyrimidine (136 g, 559.4mmol, 1 equiv.) in acetonitrile (1500 mL) followed by4,4-difluorocyclohexylamine hydrochloride (105.6 g, 615.4 mmol, 1.1equiv.) and N,N-diisopropyl ethylamine (194.88 mL, 1118.8 mmol, 2equiv). The reaction mixture was heated at 80° C. for 16 h. Thecompletion of reaction was determined by TLC and UPLC. The reactionmixture was concentrated and the residue was triturated with water (500mL). The resulting solid was filtered, washed with pet-ether, driedunder vacuum to afford6-chloro-N-(4,4-difluorocyclohexyl)-2-(3,5-dimethyl-1h-pyrazol-1-yl)pyrimidin-4-amine(191 g, 556 mmol, >95%) as an off-white solid. MS (MH+): m/z=342.0.

Step 3:

A 250 mL three-necked, flame-dried, round-bottomed flask, equipped witha teflon-coated stir bar (2 cm), one septa (neck 1), stopper (neck 3)and reflux condenser equipped with nitrogen gas inlet-outlet U-tubeadaptor filled with oil (Neck 2), was charged with a solution of6-chloro-N-(4,4-difluorocyclohexyl)-2-(3,5-dimethyl-1h-pyrazol-1-yl)pyrimidin-4-amine(20 g, 58.51 mmol, 1 equiv.) in methanol followed by sodium methoxide(21% in methanol, 5.37 g, 99.47 mmol, 1.7 equiv.). The reaction washeated to 60° C., and completion of reaction was determined by TLC andUPLC. After 5 h, the reaction mixture was concentrated under reducedpressure and the residue was diluted with ethyl acetate, washed withwater, and washed with brine solution. The organic layer was dried oversodium sulfate, filtered, and concentrated under reduced pressure toafford the crude product which was purified by column chromatographyusing ethyl acetate in pet-ether as solvent system to affordN-(4,4-difluorocyclohexyl)-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-methoxypyrimidin-4-amine(2) [16 g (11 g (99% pure)+5 g (92% pure), 47.41 mmol, ˜80%) as a whitesolid. MS (MH+): m/z=338.1. Analytical Data: ¹H-NMR (400 MHz, DMSO-d₆):δ 7.45 (bs, 1H), 6.06 (s, 1H), 5.72 (s, 1H), 4.01 (bs, 1H), 3.85 (s,3H), 2.55 (s, 3H), 2.17 (s, 3H), 2.11-1.82 (m, 6H), 1.60-1.55 (m, 2H).

N-(4, 4-difluorocyclohexyl)-6-methoxy-2-(4-methylthiazol-2-yl)pyrimidin-4-amine

Step 1:

A three-necked round bottomed flask equipped with a teflon-coated stirbar was charged with diethyl ether (250 mL) and n-BuLi (241.98 mL,604.96 mmol, 2.5M in hexane) was transferred at −78° C. A solution of4-methylthiazole (50.0 g, 504.13 mmol) in diethyl ether (200 mL) wasadded over a period of 30 min. The reaction mixture was turned into paleyellow suspension. After 1.5 hours, DMF (58.54 mL, 756.20 mmol) wasadded and stirred at room temperature for 16 h. The progress of thereaction was monitored by TLC. After completion of the reaction, themixture was poured into cold aq. HCl (400 mL, 4N) under stirring andseparated the two layers. The organic layer was washed with cold aq. HCl(2×80 mL, 4N)). The combined aq. layers were slowly basified with K₂CO₃(pH 7) and extracted with diethyl ether (3×150 mL). The combined organiclayers were dried over sodium sulfate and evaporated to dryness at roomtemperature under vacuum to afford 4-methylthiazole-2-carbaldehyde (60.0g, crude) as a pale yellow liquid. This crude material was used in thenext step without further purification.

Step 2:

A two-necked round bottomed flask equipped with a teflon-coated stir barwas charged with 4-methylthiazole-2-carbaldehyde (60.0 g, crude) inpyridine (38.04 ml, 472.40 mmol). Hydroxylamine hydrochloride (32.82 g,472.40 mmol) was added in portions over a period of 15 min. The reactionmixture was stirred at room temperature for 16 h under nitrogenatmosphere. The progress of the reaction was monitored by TLC. Aftercompletion of the reaction, the mixture was poured into ice cold waterand stirred for 20 min, the obtained solid was filtered and dried undervacuum to afford 4-methylthiazole-2-carbaldehyde oxime (40.0 g, 281.69mmol, 59% for two steps) as an off white solid. MS (MH+): m/z=143.0.

Step 3:

A two-necked round bottomed flask equipped with a teflon-coated stir barwas charged with a solution of 4-methylthiazole-2-carbaldehyde oxime(35.0 g, 246.44 mmol) and pyridine (87.33 mL, 1084.35 mmol) in 1,4-dioxane (140 mL). Trifluoroacetic anhydride (51.38 mL, 369.66 mmol)was added slowly at −10° C. and allowed to stir at room temperature for16 h. The progress of the reaction was monitored by TLC. Aftercompletion of the reaction, the mixture was diluted with water (250 mL)and extracted with diethyl ether (3×350 mL). The combined organic layerswere washed with water (2×250 mL), brine (100 mL) dried over sodiumsulphate and concentrated under reduced pressure to afford4-methylthiazole-2-carbonitrile (35.0 g, crude) as light brown liquid.This crude material was used in the next step without furtherpurification. Analytical Data: ¹H-NMR (400 MHz, DMSO-d₆): δ 7.90 (s,1H), 2.51 (s, 3H).

Step 4:

A two-necked round bottomed flask equipped with a teflon-coated stir barwas charged with 4-methylthiazole-2-carbonitrile (35.0 g, crude) inmethanol (280 mL) and sodium methoxide (16.77 g, 310.45 mmol) was added.After stirring at room temperature for 3 h, ammonium chloride (30.19 g,564.66 mmol) was added and stirred for another 16 h. The progress of thereaction was monitored by TLC. After completion of the reaction, themixture was filtered and washed with methanol. The filtrate wasconcentrated under reduced pressure and the residue was triturated withdiethyl ether (150 mL). The formed solid was filtered and dried undervacuum to afford 4-methylthiazole-2-carboximidamide hydrochloride (35.0g, crude) as an off-white solid. This crude material was used in thenext step without further purification. MS (MH+): m/z=142.0.

Step 5:

A two-necked round bottomed flask equipped with a teflon-coated stir barwas charged with 4-methylthiazole-2-carboximidamide hydrochloride (35.0g, crude) in ethanol (350 mL) and diethyl malonate (150.81 mL, 988.64mmol). Sodium ethoxide (320 mL, 988.64 mmol, 21% in EtOH) was addeddropwise at room temperature and heated to 85° C. After 3 hours, thereaction mixture was concentrated under reduced pressure. Water (20 mL)was added and acidified with 1.5 N HCl (pH 2-3). The obtained solid wasfiltered and dried under vacuum to afford 2-(4-methylthiazol-2-yl)pyrimidine-4, 6-diol (29.0 g, crude) as pale yellow solid. This crudematerial was used in the next step without further purification. MS(MH+): m/z=210.0.

Step 6:

A two-necked round bottomed flask equipped with a teflon-coated stir barwas charged with a suspension of 2-(4-methylthiazol-2-yl) pyrimidine-4,6-diol (29.0 g, crude) and POCl₃ (290 mL). N,N-diethylaniline (37.84 mL,235.85 mmol) was added at room temperature and heated reflux at 100° C.for 2 h. The progress of the reaction was monitored by TLC. Excess POCl₃was removed by distillation. The residue was diluted with 500 mL coldwater, neutralized with saturated sodium bicarbonate solution, extractedwith diethyl ether (2×500 mL). The combined organic layers were washedwith water (3×200 mL), brine (100 mL), dried over sodium sulfate andconcentrated under reduced pressure. The residue was triturated withn-pentane (100 mL). The obtained solid was filtered and dried undervacuum to afford 2-(4, 6-dichloropyrimidin-2-yl)-4-methylthiazole 7(19.5 g, 79.59 mmol, 32% for four steps) as a pale yellow solid. MS(MH+): m/z=245.9.

Step 7:

A two necked round bottomed flask equipped with a teflon-coated stir barwas charged with a suspension of2-(4,6-dichloropyrimidin-2-yl)-4-methylthiazole (19.0 g, 77.56 mmol) and4, 4-difluorocyclohexan-1-amine hydrochloride (13.30 g, 77.56 mmol) inacetonitrile (190 mL). Cesium carbonate (37.89 g, 116.34 mmol) was addedand the reaction mixture was heated at 80° C. for 16 h. The progress ofthe reaction was monitored by TLC. The reaction mixture was cooled toroom temperature, filtered, and the solid was washed with ethyl acetate(500 mL). The filtrate was washed with water (2×100 mL), brine (100 mL),dried over sodium sulfate, and concentrated under reduced pressure. Theresidue was purified by column chromatography (60-120 silica gel) elutedwith 15% EtOAc in hexane. Relevant fractions containing the requiredcompound were combined and evaporated to dryness under reduced pressureto afford6-chloro-N-(4,4-difluorocyclohexyl)-2-(4-methylthiazol-2-yl)pyrimidin-4-amine(22.5 g, 65.25 mmol, 84%) as off-white foam solid. MS (MH+): m/z=344.9.

Step 8:

A two-necked round bottomed flask equipped with a teflon-coated stir barwas charged with 6-chloro-N-(4,4-difluorocyclohexyl)-2-(4-methylthiazol-2-yl) pyrimidin-4-amine (27.0g, 78.47 mmol) in methanol (450 mL). Sodium methoxide (21.19 g, 392.36mmol) was added and heated to 80° C. for 16 h. The progress of thereaction was monitored by TLC. Excess methanol was removed under reducedpressure and the residue was diluted with 10% aqueous ammonium chloridesolution (100 mL) and extracted with ethyl acetate (3×150 mL). Thecombined organic layers were washed with water (2×100 mL), brine (100mL), dried over sodium sulphate and concentrated under reduced pressure.The residue was purified by column chromatography (60-120 silica gel)eluting with 35-40% of EtOAc in hexane. Relevant fractions containingthe target compound were combined and evaporated to dryness underreduced pressure to afford N-(4,4-difluorocyclohexyl)-6-methoxy-2-(4-methylthiazol-2-yl)pyrimidin-4-amine (3) (23.4 g, 68.82 mmol, 87%) as an off-white solid.MS (MH+): m/z=341.0. Analytical Data: ¹H-NMR (400 MHz, DMSO-d₆): δ 7.41(s, 1H), 7.40 (s, 1H), 5.81 (s, 1H), 3.87 (s, 3H), 2.43 (s, 3H),2.08-1.89 (m, 6H), 1.61-1.52 (m, 2H).

(S)-1-(6-((4,4-difluorocyclohexy)amino)-2-(4-methylthiazol-2-yl)pyrimidin-4-yl)ethan-1-ol

Step 1:

A 250-mL sealed tube, equipped with a teflon-coated stir bar (2 cm), wascharged with a solution of6-chloro-N-(4,4-difluorocyclohexyl)-2-(4-methylthiazol-2-yl)pyrimidin-4-amine(4.9 g, 14.24 mmol, 1.0 eq) and tributyl(1-ethoxyvinyl)stannane (5.65 g,15.66 mmol, 1.1 eq) in N,N-dimethylformamide (60 mL). The reactionmixture was degassed using argon gas for 5-10 min, followed by additionof bis(triphenylphosphine)palladium(II) dichloride (0.2 g, 0.28 mmol,0.02 eq). The reaction mixture was sealed and heated at 80° C. for 16 h(completion of reaction was determined by LCMS) and cooled to roomtemperature. The reaction mixture was diluted with water (300 mL) andextracted with ethyl acetate (2×150 mL). The combined organics weredried over sodium sulfate, filtered, and evaporated to afford a crudeproduct as a light brown sticky solid. The crude material was purifiedby column chromatography (ethyl acetate/hexane as solvent system) toaffordN-(4,4-difluorocyclohexyl)-6-(1-ethoxyvinyl)-2-(4-methylthiazol-2-yl)pyrimidin-4-amine(4.1 g, 10.78 mmol, 75%) as an off-white solid. MS (MH+): m/z=381.0.

Step 2:

A round-bottomed flask equipped with a teflon-coated stir bar wascharged withN-(4,4-difluorocyclohexyl)-6-(1-ethoxyvinyl)-2-(4-methylthiazol-2-yl)pyrimidin-4-amine(9.0 g, 23.67 mmol, 1.0 eq) in acetone (120 mL) followed by addition of2N hydrochloric acid aqueous solution (20 mL). The reaction mixture wasstirred at room temperature for 3 hours and completion of reaction wasdetermined by LCMS. The reaction mixture was concentrated to removeacetone, diluted with ice cold water (100 mL), basified with saturatedsodium by carbonate solution, and extracted with ethyl acetate (2×100mL). The combined organics were dried over sodium sulfate, filtered, andevaporated under reduced pressure to afford a crude product as a lightbrown sticky solid. The crude material was purified by columnchromatography (ethyl acetate/hexane as solvent system) to afford1-(6-((4,4-difluorocyclohexyl)amino)-2-(4-methylthiazol-2-yl)pyrimidin-4-yl)ethan-1-one(6.1 g, 17.32 mmol, 73%) as an off-white solid. MS (MH+): m/z=353.0.

Step 3:

A round-bottomed flask equipped with a teflon-coated stir bar wascharged with1-(6-((4,4-difluorocyclohexyl)amino)-2-(4-methylthiazol-2-yl)pyrimidin-4-yl)ethan-1-one(5.6 g, 15.90 mmol, 1.0 eq) in methanol (80 mL) at −10° C. followed bysodium borohydride (0.302 g, 7.95 mmol, 0.5 eq). The reaction mixturewas stirred at same temperature for 1 hour and completion of reactionwas determined by LCMS. The reaction mixture was quenched with water andconcentrated under reduced pressure to remove methanol. The residue wasdiluted with ice cold water (100 mL) and extracted with ethyl acetate(2×100 mL). The combined organics were dried over sodium sulfate,filtered, and evaporated under reduced pressure to afford1-(6-((4,4-difluorocyclohexyl)amino)-2-(4-methylthiazol-2-yl)pyrimidin-4-yl)ethan-1-ol4 (5.5 g, 15.53 mmol, 97%) as an off-white solid of racemic mixture. MS(MH+): m/z=355.0.

Step 4:

The racemic compound1-(6-((4,4-difluorocyclohexyl)amino)-2-(4-methylthiazol-2-yl)pyrimidin-4-yl)ethan-1-ol4 (5.5 g) was purified by chiral HPLC (Column: Chiralpak-IC(250*20*5.0μ); Mobile phase-A:N-Hexane (0.1% DEA), Mobile phase-B:IPA:DCM (90:10) isocratic: 50:50 (A:B); Flow rate: 15.0 ml/min; 120/inj;Run time: 15 min) to afford(S)-1-(6-((4,4-difluorocyclohexyl)amino)-2-(4-methylthiazol-2-yl)pyrimidin-4-yl)ethan-1-ol5 (2.1 g, 5.93 mmol, 38%) as an off-white solid from first elutingfractions (Peak-1, RT=4.24 min.). MS (MH+): m/z=355.0. ¹H NMR (400 MHz,DMSO-d₆): δ 7.59-7.57 (d, J=6.0 Hz, 1H), 7.37 (s, 1H), 6.64 (s, 1H),5.37-5.36 (d, J=4.4 Hz, 1H), 4.52-4.50 (t, J=11.2 Hz, 5.6 Hz, 1H), 4.05(bs, 1H), 2.43 (s, 3H), 2.10-1.96 (m, 6H), 1.62-1.59 (m, 2H), 1.35-1.33(d, J=6.4 Hz, 3H). Other enantiomer:(R)-1-(6-((4,4-difluorocyclohexyl)amino)-2-(4-methylthiazol-2-yl)pyrimidin-4-yl)ethan-1-ol6 (2.05 g, 5.78 mmol, 37%) as an off-white solid from second elutingfractions (Peak-2, RT=6.45 min.). MS (MH+): m/z=355.0. ¹H NMR (400 MHz,DMSO-d₆): δ 7.60-7.59 (d, J=5.6 Hz, 1H), 7.37 (s, 1H), 6.64 (s, 1H),5.38 (bs, 1H), 4.52-4.51 (d, J=6.8 Hz, 1H), 4.10 (bs, 1H), 2.43 (s, 3H),2.10-1.91 (m, 6H), 1.65-1.57 (m, 2H), 1.35-1.34 (d, J=6.8 Hz, 3H).

2-(3-cyclopropyl-1H-pyrazol-1-yl)-N-(4,4-difluorocyclohexyl)-6-morpholinopyrimidin-4-amine

Step 1:

A 1000-mL three-necked, flame-dried, round-bottomed flask, equipped witha teflon-coated stir bar (3 cm), one septa (neck 1), stopper (neck 3)and reflux condenser equipped with nitrogen gas inlet-outlet U-tubeadaptor filled with oil (Neck 2), was charged with a solution of4,6-dichloro-2-(methylthio)pyrimidine (150 g, 768.94 mmol, 1.0 equiv.)in acetonitrile (1500 mL) followed by 4,4-difluorocyclohexylaminehydrochloride (158.35 g, 922.733 mmol) and cesium carbonate (526 g, 1614mmol, 2.1 equiv.). The reaction mixture was heated at 75° C. for 16 h.The reaction mixture was filtered to remove cesium carbonate, then thefiltrate was concentrated under reduced pressure to afford 210 g (93%yield) of6-chloro-N-(4,4-difluorocyclohexyl)-2-(methylthio)pyrimidin-4-amine as apale yellow solid. MS (MH+): m/z=294.0.

Step 2:

A solution of 6-chloro-N-(4,4-difluorocyclohexyl)-2-(methylthio)pyrimidin-4-amine (60 g, 204.24mmol, 1.0 equiv.) and morpholine (35.6 mL, 408.48 mmol, 2.0 equiv.) inacetonitrile (600 mL) was heated at 85° C. in a sealed tube for 16 h.After completion of the reaction, the reaction mixture was concentrated,and the resulting residue was quenched with ice cold water. The obtainedsolid was filtered and washed with water (500 mL), hexane (250 mL),dried under high vacuum to affordN-(4,4-difluorocyclohexyl)-2-(methylthio)-6-morpholinopyrimidin-4-amineas an off-white solid (62 g, 88% yield). MS (MH+): m/z=345.2.

Step 3:

A 100-mL three-necked, flame-dried, round-bottomed flask, equipped witha teflon-coated stir bar (3 cm), one septa (neck 1), stopper (neck 3)and reflux condenser equipped with nitrogen gas inlet-outlet U-tubeadaptor filled with oil (Neck 2), was charged with a solution ofN-(4,4-difluorocyclohexyl)-2-(methylthio)-6-morpholino pyrimidin-4-amine(1 g, 2.90 mmol) in tetrahydrofuran (15 mL) followed by4-N,N-dimethylaminopyridine (0.1 g, 0.87 mmol, 0.3 equiv.),triethylamine (1.2 mL, 8.71 mmol, 3.0 equiv.) and Boc anhydride (3.16 g,14.51 mmol, 5.0 equiv.) then the reaction mixture was heated at 80° C.for 16 h. After completion of the reaction, the reaction mixture wasquenched with water and extracted with ethyl acetate (2×75 mL). Thecombined organic layer was dried over anhydrous sodium sulfate andconcentrated to afford tert-butyl(4,4-difluorocyclohexyl)(2-(methylthio)-6-morpholinopyrimidin-4-yl)carbamate as a yellow gum (1.1 g, 85%). MS (MH+):m/z=445.2.

Step 4:

A 100-mL single neck round bottom flask, connected with reflux condenserequipped with nitrogen gas inlet-outlet U-tube adaptor filled with oil,a teflon-coated stir bar (1 cm), was charged with a solution oftert-butyl(4,4-difluorocyclohexyl)(2-(methylthio)-6-morpholinopyrimidin-4-yl)carbamate(50 g, 112.47 mmol) in dichloromethane (600 mL) followed by3-chloroperbenzoic acid (m-chloroperbenzoic acid) (58.2 g, 337.42 mmol,3.0 equiv.) at 0° C. The reaction mixture was slowly warmed to rt andstirred for 30 min. After the completion of the reaction, the reactionmixture was quenched with saturated bicarbonate solution and extractedwith dichloromethane (2×250 mL). The combined organic layer was driedover anhydrous sodium sulfate and concentrated to afford tert-butyl(4,4-difluorocyclohexyl)(2-(methylsulfonyl)-6-morpholinopyrimidin-4-yl)carbamateas an off-white gum (52 g, 97% yield). MS (MH+): m/z=477.3.

Step 5:

A 100-mL single neck round bottom flask, connected with reflux condenserequipped with nitrogen gas inlet-outlet U-tube adaptor filled with oil,a teflon-coated stir bar (2 cm), was charged with a solution oftert-butyl(4,4-difluorocyclohexyl)(2-(methylsulfonyl)-6-morpholinopyrimidin-4-yl)carbamate(0.9 g, 1.88 mmol) in acetonitrile (10 mL) followed by3-cyclopropyl-1H-pyrazole (0.3 g, 2.83 mmol, 1.5 equiv.) and cesiumcarbonate (1.23 g, 3.77 mmol, 2.0 equiv.). The reaction mixture washeated at 80° C. for 16 hours, and completion of reaction was determinedby TLC and LCMS. The reaction mixture was filtered and the filtrate wasconcentrated. The crude product was purified through columnchromatography using 60-120 silica gel with ethyl acetate-pet ether assolvent system. The isolated material was dried under vacuum to affordtert-butyl(2-(3-cyclopropyl-1H-pyrazol-1-yl)-6-morpholinopyrimidin-4-yl)(4,4-difluorocyclohexyl)carbamateas an off-white solid (0.8 g, 84%). MS (MH+): m/z=505.

Step 6:

A 100-mL three-necked, flame-dried, round-bottomed flask, equipped witha teflon-coated stir bar (2 cm), one septa (necks 1), stopper (neck 3)and nitrogen gas inlet-outlet U-tube adaptor filled with oil (Neck 2),was charged with a solution tert-butyl(2-(3-cyclopropyl-1H-pyrazol-1-yl)-6-morpholinopyrimidin-4-yl)(4,4-difluorocyclohexyl)carbamate(1.2 g, 1.98 mmol, 1 eq) in dichloromethane (40 mL) followed bytrifluoroacetic acid (2.5 mL, 32.55 mmol, 16.4 eq) at 0° C. The reactionmixture was slowly warmed to rt and stirred at same temperature for 6hours. The completion of reaction was determined by TLC and UPLC. Thereaction mixture was concentrated and the resulting residue was quenchedwith 10% saturated sodium bicarbonate solution, extracted with ethylacetate (2×100 mL), and concentrated under reduced pressure to affordcrude product. The crude product was purified through columnchromatography using 60-120 silica gel, ethyl acetate-pet ether assolvent system. The resulting solid was dried under vacuum to afford2-(3-cyclopropyl-1H-pyrazol-1-yl)-N-(4,4-difluorocyclohexyl)-6-morpholinopyrimidin-4-amine7 (0.73 g, 90%). MS (MH+): m/z=405. Analytical Data: ¹H-NMR (400 MHz,DMSO-d₆): δ 8.39 (d, J=2.4 Hz, 1H), 7.08 (d, J=8.0 Hz, 1H), 6.14 (d,J=2.80 Hz, 1H), 5.53 (s, 1H), 3.88 (s, 1H), 3.69-3.67 (m, 4H), 3.50 (m,4H), 1.99-1.90 (m, 7H), 1.56-1.54 (m, 2H), 0.93-0.89 (m, 2H), 0.72-0.71(m, 2H).

Biological Assays

The biological activity was determined as follows. The ionic currentthrough small-conductance Ca²⁺-activated K⁺ channels (SK channels,subtype 2) was measured using the whole-cell configuration of thepatch-clamp technique in a patch-clamp set-up using HEK293 tissueculture cells expressing SK2 channels as described in Hougaard et al.,British Journal of Pharmacology 151, 655-665, May 8, 2007, the entireteachings of which are incorporated herein by reference. In one aspect,a compound is defined to be an SK Positive Allosteric Modulator (PAM) ifthe compound increases current in this assay, for example, if the SC₁₀₀value of the compound is less than or equal to 10 μM as determined bythis assay. The SC₁₀₀ value is defined to be the concentration ofcompound that increases the basal current by 100%.

The SC₁₀₀ value is given in Table 1.

Male Sprague Dawley rats were administered with either Vehicle, 10, or30 mg/Kg Compound 1 by oral administration 30 minutes prior to harmalineinjection to investigate the therapeutic effect of Compound 1 onharmaline induced tremor. Immediately following harmaline injection,animals were placed in the tremor quantification apparatus and tremorevents were quantified for 60 minutes. A tremor event signal wasgenerated when a small metal transmitter band fitted to the rightforepaw of the animal moved within the electromagnetic field generatedby a loop antenna within the testing apparatus. Outputs from theamplifier were digitized at a sampling rate of 1,000 Hz and the signalwas processed and analyzed using LabView software (NationalInstruments). To minimize signal from ambulatory and grooming behavior,the signal was filtered with a 128-ms unweighted moving average filter,and events with amplitudes>0.5 V and lasting>300 ms in duration werecounted as a tremor event. Data were analyzed in one-minute bins overthe course of the test and presented as the sum of tremor events overthe entire 60 minute test. As shown by FIG. 1, significant inhibition oftremors was observed at a dose of 30 mg/Kg Compound 1.

Reduction of tremor with Compound 1 has also been demonstrated bymeasurement of whole-body tremor frequency via a force-plateaccelerometer.

Whole body tremor was measured by a San Diego Instruments Tremor Monitor(San Diego, Calif., USA). Animals were pre-treated with 3, 10, or 30mg/kg Compound 1 orally 30 minutes prior to intraperitonealadministration of 5 mg/kg harmaline. Tremor was measured for 30 minutesfollowing harmaline administration, and data were analyzed by fastFourier transform and reported as a frequency power spectrum. Harmalineinduced a significant increase in the power spectrum in a band offrequencies between 10 and 14 Hz. In this range, 3, 10, and 30 mg/kg allsignificantly reduced tremor. Data were further analyzed by calculatingthe percent Motion Power (% MP), defined as the power in the 9-13 Hzband divided by the total power across the spectrum (0-30 Hz) multipliedby 100. By this analysis, 3, 10, and 30 mg/kg significantly reducedharmaline-induced tremor (harmaline+vehicle (n=13); harmaline+3 mg/kgCompound 1 (n=8), P<0.01; 10 mg/kg Compound 1 (n=16) and 30 mg/kgCompound 1 (n=13), respectively, P<0.05) (FIG. 2).

Taken together, these data show that Compound 1 significantly reducesharmaline-induced tremor measured by two different experimental designs.

The extent to which compounds modulate SK2 channels in vivo is expressedas % SK2 SC₁₀₀, which is the ratio of the concentration of the drug freein the brain to the measured potency of the compound on the SK2 channel.It is calculated as follows: C_(FB)=C_(MB)×BFF, where C_(MB) is theconcentration of compound measured by liquid chromatography massspectrometry from brains harvested immediately following tremorrecording (Table 1, “Measured Brain Concentration”). C_(FB) is theamount of free compound not complexed with protein and therefore free tointeract with the SK2 channel (Table 1, “Calculated Free BrainConcentration”). BFF is average free fraction of compound as measured byequilibrium dialysis in separate experiments (1 uM test concentrationincubated in 10% rat brain tissue homogenate for 5 hours at 37° C.)(Table 1, “Brain Free Fraction”). Free drug in brain available tointeract with SK2 channels (C_(FB)) is arrived at by multiplying themeasured total brain level (C_(MB)) by the average free fraction (BFF).

The amount of free compound is then expressed in terms of its potencyagainst the SK2 channel as follows: % SK2 SC₁₀₀=C_(FB)/SK2 SC₁₀₀×100,where SK2 SC₁₀₀ (Table 1, “SK2 SC₁₀₀”) is the measured value of potencyof the compound on SK2 channels and % SK2 SC₁₀₀ (Table 1, “% SK2 SC₁₀₀”)is the free brain concentration (C_(FB)) normalized to SK2 SC₁₀₀. Valuesare given in Table 1.

TABLE 1 Minimally Measured Calculated Measured Efficacious BrainMeasured Free Brain SK2 Dose Concentration Brain Free ConcentrationSC₁₀₀ Calculated % Compound (mg/Kg) (μM) Fraction (μM) (μM) SK2 SC₁₀₀ 130 1.3 0.065 0.08 0.5 16

Effect on Purkinje Cell Firing Irregularity in Ex Vivo Slices from SCA258Q Transgenic Mice

In cerebellar slices from SCA2 58Q mice, Purkinje neurons exhibitchaotic firing, measurable as an increase in the coefficient ofvariation of the interspike interval (ISI CV), a measure of theregularity of the firing interval between action potentials. Thedifference in ISI CV between wild-type (N=8) and SCA2 58Q (N=11) mice isillustrated in FIG. 3 (P<0.005). Also shown, sequential bath applicationof 1 (N=11) or 3 μM (N=10) Compound 1 partially reversed the increase inISI CV observed in cerebellar slices from eleven-month old SCA2 58Q mice(P<0.05). These data indicate that Compound 1 regularizes Purkinjefiring by partially restoring the interspike interval in this mousemodel of Spinocerebellar Ataxia.

Evaluation of Compound Effect in Episodic Ataxia 2 (EA2) Tottering MouseModel

Compound 1 has demonstrated efficacy in validated animal models ofhereditary ataxia (EA2) and ET (harmaline-induced tremor). To testwhether Compound 1 can alleviate ataxia in a disease model, it wasevaluated in the EA2 “Tottering” mouse model. These mice display a basalataxia that arises from irregularity in Purkinje cell pacemaker firingdue to a loss-of-function mutation in P/Q Ca²⁺ channels (the sameprotein that is mutated in SCA6), which causes Episodic Ataxia 2 (EA2).Animals were assessed in the parallel rod floor apparatus whichautomatically counts the number of times the animal's foot slips throughthe evenly spaced metal rods and the total distance the animal travels.Baseline ataxia was then expressed as the Ataxia Ratio, which is thetotal number of foot slips divided by the total distance the animaltravels in centimeters. The increase in Ataxia Ratio between wild-typeand EA2 mice is illustrated in FIG. 4.

In this study, EA2 (8-10 months old; n=18) mice were injectedintraperitoneally with Compound 1 or vehicle 30 minutes prior to beingplaced in the parallel rod floor apparatus. Animals were assessed in across-over study design with each animal receiving both vehicle and 10mg/kg Compound 1. Three days were allowed between doses for washout ofthe compound. At the dose administered in this study, Compound 1 fullyreversed the increase in Ataxia Ratio observed in EA2 vs wild-type mice.These data indicate that Compound 1 restores normal performance in thismeasure of motor function in a model of hereditary cerebellar ataxia.

Comparative Advantages

The following studies illustrate further technical advantages of thecompounds disclosed herein.

Aqueous solubility (kinetic solubility) tests of the compounds wereperformed in phosphate buffer saline (pH 7.4) measured by shake-flaskmethod. In this assay, DMSO stock solution of test compound is added tobuffer followed by equilibration (shaking), filtration and determinationof soluble amount by HPLC-UV Conditions used in the assay are summarizedbelow. Results are shown in Table 2 and Table 3.

-   -   Compound concentration: 200 μM with 1% DMSO (n=2)    -   Aqueous buffer: 0.05M Phosphate Buffer System pH 7.4    -   Equilibration period: 16 hours at room temperature (˜23° C.)        with agitation    -   Sample preparation: Filtration    -   Analysis: HPLC-UV    -   Reference compounds: Caffeine (high solubility) and        Diethylstilbestrol (low solubility)

Metabolic stability profiling of the compounds was performed in livermicrosomes. In this assay, test compound is incubated with livermicrosomes in the presence of NADPH for 2 time points at 37° C. At theend of incubation, reaction is quenched with acetonitrile containinginternal standard and the parent compound remaining is determined byLC-MS/MS. Conditions used in the assay are summarized below. Results areshown in Table 2.

-   -   Incubation time: 0 and 30 minutes at pH 7.4, 37° C.    -   Test concentration: 1 μM in 0.02% DMSO at pH 7.4 (n=2)    -   NADPH concentration in assay: 1 mM    -   Liver microsome protein concentration in assay: 0.5 mg/ml    -   Analysis: LC-MS/MS    -   Data reported: % Parent Compound Remaining (% PCR)    -   Reference compounds for high and low clearance are included    -   Species tested: mouse, rat, dog, monkey, and human

Compounds described herein were tested for CYP inhibition across 5isoforms (3A4, 2D6, 2C9, 2C19 & 1A2). In this assay, CYP isoformspecific substrates are incubated with human liver microsome (HLM) inthe presence of test compounds, and metabolite formation is determined.Percentage inhibition of metabolite formation at differentconcentrations of test compound is calculated and IC₅₀ is determined.Conditions used in the assay are summarized below. Results are shown inTable 2.

-   -   Test drug (Inhibitor) Concentration: 8 different concentrations        (100 μM to 0.005 μM)    -   Matrix: Human Liver Microsome (Invitrogen, life technologies)    -   Specific probe substrates will be used for the isoforms as given        below:    -   CYP3A4: Midazolam    -   CYP2D6: Bufuralol    -   CYP2C9: Diclofenac    -   CYP2C19: Mephynitoin    -   CYP1A2: Phenacetin    -   Cofactors: NADPH (1 mM final in assay)    -   Sample Analysis: LC-MS/MS (metabolites)    -   Specific reference inhibitors included in all assays        (Ketoconazole/Quinidine/Sulphaphenazole/Ticlopidine/Furafylline)    -   Buffer: Potassium Phosphate Buffer (100 mM) pH 7.4    -   DMSO level in assay: 0.1%    -   Data analysis: % Inhibition over control

Compounds were tested in a cardiac potassium channel hERG assay. hERG isresponsible for a rapid delayed rectifier current (I_(Kr)) in humanventricles. Inhibition of I_(Kr) is the most common cause of cardiacaction potential prolongation by non-cardiac drugs. See e.g., Brown, A.M. and Rampe, D. (2000). Drug-induced long QT syndrome: is HERG the rootof all evil? Pharmaceutical News 7, 15-20.

HEK-293 cells were stably transfected with hERG cDNA. Stabletransfectants were selected by coexpression with the G418-resistancegene incorporated into the expression plasmid. Selection pressure wasmaintained by including G418 in the culture medium. Cells were culturedin Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (D-MEM/F-12)supplemented with 10% fetal bovine serum, 100 U/mL penicillin G sodium,100 μg/mL streptomycin sulfate and 500 μg/mL G418. Cell culture recordsare kept on file at Charles River Laboratories.

Cells were transferred to the recording chamber and superfused withvehicle control solution. Pipette solution for whole cell recordings was(composition in mM): potassium aspartate, 130; MgCl₂, 5; EGTA, 5; ATP,4; HEPES, 10; pH adjusted to 7.2 with KOH. Pipette solution was preparedin batches, aliquoted, stored frozen, and a fresh aliquot thawed eachday. Patch pipettes were made from glass capillary tubing using a P-97micropipette puller (Sutter Instruments, Novato, Calif.). A commercialpatch clamp amplifier was used for whole cell recordings. Beforedigitization, current records were low-pass filtered at one-fifth of thesampling frequency.

Onset and steady state inhibition of hERG current was measured using apulse pattern with fixed amplitudes (conditioning prepulse: +20 mV for 2s; test pulse: −50 mV for 2 s) repeated at 10 s intervals, from aholding potential of −80 mV. Peak tail current was measured during the 2s step to −50 mV.

One test article concentration was applied to each cell (n=3). Peakcurrent was measured during the test ramp. A steady state was maintainedfor at least 30 seconds before applying test article or positivecontrol. Peak current was measured until a new steady state wasachieved. Results are shown in Table 2.

Oral bioavailability data was collected in rat as follows. Results areshown in Table 2.

-   -   Rat strain/sex: Sprague Dawley/Male    -   Age/body weight: 6 to 8 weeks/250-300 gms    -   No of animals per group: n=3    -   Total no of groups: 2 (1 mpk IV & 10 mpk PO)    -   Route of administration: Oral (PO)/Intravenous (IV)    -   Dosing volume: Intravenous (2 ml/kg) & Oral (10 ml/kg)    -   Formulation vehicles: Standard formulations or suggested by        Sponsor Dose levels (IV & Oral): 1 mg/kg; intravenous & 10        mg/kg; oral or as suggested    -   Fast/Fed: Oral dosing will be performed with overnight fasted        animal Dosing frequency: Single dose    -   Time points for blood collection: (57 plasma samples for        analysis) IV—10 points (pre-dose; 5 min; 15 min; 30 min; 1 h; 2        h; 4 h; 6 h; 8 h; 24 h) [n=3 rats]    -   PO—9 points (pre-dose; 15 min; 30 min; 1 h; 2 h; 4 h; 6 h; 8 h;        24 h) [n=3 rats]    -   Blood samples collection: Jugular vein cannula    -   Anti-coagulant: 0.2% K2 EDTA    -   Sample analysis by discovery grade bioanalytical method        developed for estimation of test compound in plasma using        LC-MS/MS systems.

TABLE 2

  Compound 1

  Comparator A

  Comparator B SK2 potency (SC₁₀₀)^(a) 400 nM 1380 nM  5800 nM BrainFree Fraction^(a) 6.1%  1.2%  NA Kinetic solubility 0.074 0.017 NA(mg/mL) Liver Mouse 47%  1% NA microsome Rat 60%  1% NA stability^(†)Dog 54%  2% NA Monkey 46%  1% NA Human 84% 50% NA Cyp1A2 inhibition >10uM 640 nM NA (IC₅₀) Cyp3A4 inhibition >10 uM >10 uM NA (IC₅₀) Cyp2D6inhibition >10 uM 790 nM NA (IC₅₀) hERG inhibition >10 uM >10 uM NA(IC₅₀) Oral bioavailability^(§) 81%  5% NA SC₁₀₀ = Concentration thatproduces doubling of channel current ^(a)Small discrepancy in numberwhen compared with prior table due to insignificant differences inaverages taken from later experiments. ^(†)% remaining at 1 hour ^(§)10mg/kg PO, 0.5 mg/kg IV in rat

As shown by the data in Table 2 above, Compound 1 is over 3-fold morepotent on SK2 then Comparator A and over 14-fold more potent on SK2 thenComparator B. Compound 1 also has better solubility, higher BFF, highermicrosomal stability, and greater oral bioavailability than ComparatorA. An overall improvement in BFF and solubility over Comparator A andphenyl derivative Comparator C was also demonstrated across thecompounds described herein as shown in Table 3. Compound 1 is reproducedfrom above for ease of comparison.

TABLE 3 Kinetic Brain Free solubility Fraction (mg/mL)

  Comparator C 0.16% 0.001 

  Compound 1  6.1% 0.074 

  Compound 2 2.76% 0.031 

  Compound 3 2.69% 0.0709

  Compound 5 4.20% 0.0803

  Compound 6 4.31% 0.0667

  Compound 7 1.55% 0.0497

While we have described a number of embodiments of this invention, it isapparent that our basic examples may be altered to provide otherembodiments that utilize the compounds and methods of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims rather than by the specificembodiments that have been represented by way of example.

The contents of all references (including literature references, issuedpatents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein in their entireties by reference. Unless otherwisedefined, all technical and scientific terms used herein are accorded themeaning commonly known to one with ordinary skill in the art.

The invention claimed is:
 1. A method of treating a disease or conditionselected from ataxia, dystonia, tremors, Parkinson's disease, ischemia,traumatic brain injury, amyotrophic lateral sclerosis, hypertension,atherosclerosis, diabetes, arrhythmia, over-active bladder, anxiety,epilepsy, insomnia, and withdrawal symptoms caused by the termination ofabuse of alcohol and other drugs of abuse in a subject in need thereofcomprising the step of administering to the subject a compound of theformula:

or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein the disease or condition is anxiety.
 3. The method of claim 1,wherein the disease or condition is Parkinson's disease.
 4. A method oftreating essential tremor in a subject in need thereof comprising thestep of administering to the subject a compound of the formula:

or a pharmaceutically acceptable salt thereof.
 5. A method of treatingataxia in a subject in need thereof comprising the step of administeringto the subject a compound of the formula:

or a pharmaceutically acceptable salt thereof.
 6. The method of claim 5,wherein the ataxia is spinocerebellar ataxia.